Abstract

Background & Aim Mesenchymal stromal cells (MSCs) have been shown to exert important immunomodulatory effects in both acute and chronic diseases. In acute inflammatory conditions, an “off-the-shelf” cryopreserved, allogeneic cell product might be better suited as it can be ready for administration to patients without delay. Genetically modified cells have recently gained tremendous interest given the successes in chimeric antigen receptors (CAR T) cell therapy, in which T cells from patients can be genetically engineered to produce an artificial T-cell receptor for treating cancer. However, whether a genetically modified, allogeneic MSC can have sustained transgene expression post cryopreservation has not yet been well studied. Herein we assess the effect of non-viral transfection on cell viability and protein expression in cryopreserved-and-then-thaw MSC. Methods, Results & Conclusion MSCs were derived from bone marrow of healthy donors and characterized using the ISCT criteria (surface marker expression and tri-lineage differentiation potentials). MSCs were transfected with non-viral plasmid using two protocols: 1) transfection 24 hours after cell seeding, or 2) transfection after 6 days of culture. Transfected MSCs were cryopreserved at 24 hours after transfection. Viability (Trypan blue), cell number recovery and transgene expression (Western blot) were examined. Prior to cryopreservation, the cell viability was 87% ± 3% from protocol 1 (n=2) and 93% ± 1.2% from protocol 2 (n=3). After one week of storage in liquid nitrogen, cell viability was 71% ± 4% and 82% ± 5% for Protocol 1 and 2 respectively, while the percentage of cell recovery post thaw was 72% ± 9% and 95% ± 2%. Thawed cells were sub-cultured to confirm no abnormality in cell growth and morphology. To determine levels of transgene protein expression, cell pellets were collected pre- and post-cryopreservation. Western blot analysis showed that `transgene protein could be detected 2 days after cells were thawed, and the protein expression was modestly reduced to 78% ± 6.7% to that of pre-cryopreservation in protocol 1 and 93%± 1.7% in protocol 2. Our results address the concerns that transgene expression might be lost during the process of cryopreservation by showing a sustained but slightly reduced level of proteins overexpression to that of pre-cryopreservation. Further optimization will be conducted to minimize the loss of protein expression and to improve viability after thaw.

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